Although it is well known that sickle cell disease is caused by a genetic disorder of hemoglobin (Hb S), the mechanisms of initiation of vaso-occlusive crisis as well as the formation of dense cells and irreversibly sickled cells (ISCs) are unknown. Most past experiments concerning sickling of SS cells have been done under completely deoxygenated conditions, while SS cells in vivo exist under dynamic deoxygenation-oxygenation (d-o) cycle conditions. Recently I built an apparatus with which SS cells can be exposed to repeated d-o cycles mimicking conditions in vivo. Using this apparatus I found that most SS cells assumed a granulated and star-shaped morphology after the first deoxygenation, but then gradually changed to the typical elongated sickled shape during successive d-o cycles. Preliminary results showed that SS cells with an elongated sickle shape under deoxygenated conditions are more dehydrated than SS cells with a maple-leaf or star-shape morphology. These findings suggest that the formation of dense cells and ISCs is closely related to the morphology of deoxygenated SS cells. The purpose of this project is to investigate the relationship between changes in morphology of SS cells during d-o cycles and the formation of dense cells and ISCs. Our hypothesis is that formation of dense cells and ISCs is directly related to changes in membrane properties that can cause abnormal ion movement and induce dehydration. We also hypothesize that a critical degree of deformation or elongation must be reached before SS cells become dehydrated and irreversibly deformed. For precise evaluation of changes in shape of SS cells, we will use an automated image analysis system. Although the classic visual method gives us information about various morphologies, this image analysis system allows us to evaluate numerically the shape of deoxygenated and oxygenated SS cells. I also found that oxygen dissociation curves (ODCs) and oxygen association curves (OACs) of SS cells shifted progressively to the fight during d-o cycles. No shifts in ODCs and OACs were observed for normal (AA) cells. These alterations are thought to be caused by an increase in Hb S polymer fraction and/or by the increase in intracellular hemoglobin concentration (MCHC) due to dehydration. These results suggest again that the formation of sickled cells is closely related to oxygen binding properties and formation of dense cells and ISCs during d-o cycles. We will investigate the relationship between morphology and the formation of dense cells and ISCs under conditions similar to those in vivo. Effects of Ca++ and ion-flux inhibitors on membrane permeability to Na+, K+, and Cl- will also be studied. We believe that this project will provide new insights into the mechanism of initiation of vaso-occlusive crisis and formation of dense cells and ISCs.